Zika virus (ZIKV) is an urgent global health concern with epidemics in Latin America and U.S. territories that have precipitated a rise in fetal neurodevelopmental abnormalities. It is well established that neurotropic viruses such as cytomegalovirus (CMV) can cause a range of sequelae in the infected human fetus and infant, yet we know little about the trafficking of such teratogenic viruses during critical periods of developmental susceptibility. The overriding objective of this application is to develop reliable methods to label and track ZIKV in order to assess the location of the virus in real-time. Positron emission tomography (PET) is the most specific and sensitive method for imaging and assessing molecular interactions in the human body. Thus, these studies will focus on PET imaging with correlative computed tomography (CT) for virus detection and quantification through the following Specific Aims: (1) Adapt our established methods for radiolabeling to ZIKV and apply these techniques in vivo during fetal infection. We will adapt our PET/CT protocols that we have previously utilized during pregnancy including zirconium-89 (89Zr) and an antibody that targets the ZIKV envelope protein. Studies will include synthesis, in vitro assessments for sensitivity, and in vivo testing after direct fetal inoculation in utero. (2) Monitor ZIKV transplacental trafficking after maternal infection using PET/CT. In these studies we will track ZIKV after maternal inoculation at different gestational time points, and determine the temporal severity of disease as it relates to trafficking of ZIKV through the placenta to the developing fetal brain. This R21 proposal will develop state-of-the-art innovative in vivo imaging methods that will be integrated with anatomical and molecular assessments of fetal developmental outcomes. These investigations will provide new insights into viral trafficking and potential viral reservoirs in the maternal, placental, and fetal compartments, and aid in developing new strategies to protect the fetus from congenital disease.